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RAPID COMMUNICATION
Fibrinogen y-Chain mRNA Is Not Detected In Human Megakaryocytes
By Winand Lange, Andreas Luig, Gottfried Dolken, Roland Mertelsmann, and Lothar Kanz
Human megakaryocytes and platelets contain counterparts
of several plasma proteins. The origin of most of these
a-granule proteins is unclear. Fibrinogen represents one of
those molecules, being essential in hemostasis, thrombosis,
and platelet aggregation. To study whether fibrinogen is
endocytosed by megakaryocytes and packaged into a-granules or newly synthesized by these cells, w e established a
highly sensitive nested primer polymerase chain reaction for
the detection of human fibrinogen y-chain mRNA. In enriched
megakaryocyte fractions, as well as fluorescence-activated
cell sorter-purified megakaryocytes from bone marrow samples of healthy volunteers, no fibrinogen rchain mRNA could
be detected, despite the presence of the corresponding
fibrinogen y-chain DNA. We conclude that fibrinogen rchain
mRNA, as detectable by our amplification system, is missing
in megakaryocytes. This finding suggests that fibrinogen
might be acquired from plasma by endocytosis and sequestered in a-granules before reentering the circulation after
platelet activation.
0 1991 by The American Society of Hematology.
F
after written informed consent, with the approval of the Ethical
Review Committee at the University of Freiburg, Germany. Bone
marrow cells were aspirated directly into 20-mL syringes containing 1/10 vol ACD, EDTA, and prostaglandin E, and processed as
previously described.’ ’” The cell suspension was filtered, diluted
with a solution containing sodium-citrat, theophyllin, bovine serum
albumin and glucose (MK-medium), adjusted with Percoll (Pharmacia Fine Chemicals, Freiburg, Germany) to a final density of
1.020 g/mL, layered over Percoll of density 1.055 g/mL, and finally
overlayed with MK-medium. After centrifugation (400g, 20 minutes) megakaryocytic cells could be recovered from the upper
Percoll layer and the interface (density < 1.055 gimL). For fluorescence-activated cell sorter (FACS) analysis and sorting, the cells
were labeled with saturating concentrations of fluorescinated
monoclonal antibody IOP61 (Dianovaihmunotech, Hamburg,
Germany) directed against glycoprotein lIIa (anti-GPIIIa: CD61)
and analyzed with a FACS 440 System equipped with a 90-pm
nozzle (Becton Dickinson, Sunnyvale, CA). Control cells were
labeled with mouse IgG1-fluorescein isothiocyanate. GPIIIa positive cells were sorted under sterile conditions at a rate of about 500
cellslsecond into 1.5-mL test tubes. RNA extraction immediately
followed the sorting of cells. Routinely, some cells were sorted onto
small areas of a glass slide” to control for fluorescent cells by
fluorescence microscopy.
Control cells. As controls we used the fibroblast cell line MRC 5
and the hepatoma cell line HepG2. Fibroblasts as well as HepG2
cells were grown in Eagle’s minimal essential medium containing
10% fetal calf serum, 15 mmol/L Tricine buffer (Sigma, Deisenhofen, Germany), and penicillinistreptomycin.
RNA isolation. Total RNA was isolated from freshly prepared
megakaryocytes or cultured cells by the acid guanidinium thiocyanate-phenol-chloroform extraction as described by Chomczynski
and Sacchi.” Five micrograms of yeast tRNA was added to 2 X lo6
cells as carrier RNA to facilitate RNA isolations. RNA samples
were divided into multiple aliquots for further analysis.
In vitro amplification. PCR was performed as previously de~cribed,’~
with minor modifications. Oligonucleotides were chemically synthesized and purified on oligonucleotide purification
cartridges (Applied Biosystems, Foster City, CA). The sequence
information to synthesize the primers and probes was taken from
the published literature.l4.”
For the amplification of fibrinogen y-chain mRNA the following
primers were used: F1 (external sense, position 1813 to 1834):
5’-GCACCCCCGGAATTTAATTCTC-3’;
F2 (internal sense, position 1981 to 2002): 5’-TACCAGAGACAACTGCTGCATC-3’;
F3 (internal a-sense, position 2243 to 2222): 5‘-AATCTGCAATGCCACAGGTAGT-3’; F4 (external a-sense, position 2310 to
2289): 5’-ACTTGATGTAAGATGTCTTCCA-3’.
For GPIIIa the primers were: G1 (external sense, position 1116
to 1138): S’-GTCCTCCAGCTCATTGTGATGC-3’; G2 (inter-
IBRINOGEN IS essential for fibrin formation and
platelet aggregation. Human fibrinogen consists of
three pairs of nonidentical polypeptide chains, each encoded by separate genes, clustered on chromosome 4q.’
The interaction with the GPIIb-IIIa heterodimer receptor
on platelets occurs through two different binding sites on
the fibrinogen molecule, located on the a-chain (RGDsequences)’ and the y-chain (d~decapeptide).~
The carboy
terminus of the y-chain also represents the site involved in
factor XIIIa-mediated crosslinking of fibrim4Soluble fibrinogen represents 2% to 3% of the plasma proteins and is
mainly derived from hepatocytes; its synthesis is directly
stimulated by interleukin-6 (hepatocyte-stimulating factor)
through increasing transcriptional activity of these cells5
An additional small pool is known to exist in a-granules of
bone marrow megakaryocytes and circulating platelets.6
There is a long-standing controversy whether this protein,
as well as other counterparts of plasma proteins that are
found in a-granules such as von Willebrand factor, vitronectin, fibronectin and others, are synthesized by the megakaryocytes or could be acquired from the plasma and
delivered to a-granules. To study this question we analyzed
fibrinogen y-chain mRNA expression by a highly sensitive
nested polymerase chain reaction (PCR). Our results indicate that fibrinogen is not synthesized by megakaryocytes
but originates from plasma and is stored in the a-granules,
as indicated by other studie~.’,~
MATERIALS AND METHODS
Cell preparation and Jlow cytometry. Bone marrow aspirates
were obtained from the posterior iliac crest of healthy volunteers
From Department Medicine I, Hematology and Oncology, AlbertLudwigs UniversityMedical Center, Freiburg, Germany.
Submitted January 24,1991; acceptedApril I , 1991.
Supported by Deutsche Forschungsgemeinschaft, Grant No. Ka
58511-4.
Address reprint requests to Lothar Kanz, MD, Innere Medizin t
Hamatologie u. Onkologie, Medizinische Universitatsklinik Freiburg,
Hugstetter Str. 55, 0-7800 Freiburg, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 I991 by The American Society of Hematology.
0006-497119117801-0032$3.OO/O
20
Blood, Vol78, No 1 (July 1). 1991: pp 20-25
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LACK OF FIBRINOGEN EXPRESSION IN MEGAKARYOCYTES
nal sense, position 1378 to 1400): 5’-TCCAGGTCACCTTTGATTGTGAC-3’; G3 (internal a-sense, position 1680 to 1659): 5’AGGAGAAGTCGTCACACTCGCA-3’; G4 (external a-sense,
position 220 1 to 2 177): 5’-CATCACTGAGAGCAGGACCACCAGG-3’.
For aldolase A we used as primers”: A25 (exon Cl): 5’CAGCTCCTTCTTCTGCTGCGGGGTC-3’(antisense primer);
H, (exon H): 5‘-CGCAGAAGGGGTCCTGGTGA-3’
(sense
primer).
First a cDNA was synthesized with AMV reverse transcriptase
(Pharmacia) in a 75-pL reaction mixture that contained 10 mmom
KCl, 10 mmoVL MgCl,, 50 mmoVL Tris/HCI pH 8.3, 10 mmoliL
NaCl, 150 FmoliL of each deoxynucleotide triphosphate (dNTP),
60 pmol of each of the external primers, and 0.1 to 1 Fg of total
RNA. The PCR assays for the detection of the different mRNAs
were set up in separate tubes under exactly the same conditions.
They were heated at 94°C for 7 minutes followed by a 30-minute
incubation at 37°C in the presence of 5 U of reverse transcriptase.
After the addition of 5 U of Taq DNA polymerase (Perkin Elmer
Cetus, Uberlingen, Germany) the cDNA was amplified by means
of an automated thermal cycler during 30 amplification cycles: 1
minute at 92°C(denaturation),1 minute at 55°C (annealing), and 3
minutes at 72°C (extension). After 30 cycles a 5-pL aliquot served
as template for the second (nested) PCR under the same conditions as the first, except that 60 pmol of each of the internal primers
was used. In the final cycle the samples were kept at a temperature
of 72°C for 10 minutes and then chilled to 4°C.
By pretreatment of RNA templates with 2 U of RNAase-free
DNAase 1 (Pharmacia) or DNAase-free RNAase A (Pharmacia)
at a concentration of 10 pg/mL, it was possible to amplify only
RNA- or DNA-specific PCR products.
Analysis of amplified products. PCR products of the fibrinogen
y-chain and the GPIIIa PCR were analyzed only after the second
nested PCR, whereas aldolase A products were already analyzed
after one round of PCR. Aliquots of 10 p,L of the PCR mixtures
were separated on 2% or 3% agarose gels under standard conditions, the gels stained with ethidium bromide, and photographed.
The correct size of the amplified products proved the faithful
amplification of the different mRNAs; the plasmid 4x174 digested
with the restriction enzyme Hae 111 served as the molecular weight
marker. Additional restriction enzyme digests were performed
with DdeI for fibrinogen y-chain products and BamHI for GPIIIa
products generating fragments of characteristic size; reactions
were performed according to the manufacturer’s (Pharmacia)
instructions.
Yeast tRNA and primers without a template served as negative
controls for amplificationsof y-fibrinogen, GPIIIa, and aldolase A
mRNA.
RESULTS
Experiments to study fibrinogen y-chain mRNA expression in human megakaryocytes were performed with bone
marrow samples from seven healthy individuals. Percoll
density fractionation resulted in the depletion of greater
than 95% of total marrow cells and an increase in megakaryocyte frequency from about 0.05% to 3% to 7%
(4.2 +- 2.8; n = 5). In two individuals, the enriched megakaryocytes were further purified by FACS, resulting in
highly purified cells (99.1% and 98.3%, respectively). The
megakaryocytes isolated displayed all stages of cytoplasmic
and nuclear maturation. However, the most immature
megakaryocytic cells, the pro-megakaryoblasts, only numbered 2.5% to 5% of all megakaryocytes isolated.
21
There were no differences in the results of the nested
primer PCR for fibrinogen y-chain when either an enriched
cell fraction or highly purified megakaryocytes were studied.
The setup for a nested primer PCR for human fibrinogen
y-chain mRNA and DNA was chosen so that the pair of
external primers as well as the internal pair spanned several
exons and introns. The sequences of primers F1 and F4
corresponded to exon 1and exon 3 of the fibrinogen y-chain
gene generating a fragment of 498 bp, including two introns
at the DNA level and a fragment of 213 bp at the RNA
level. The internal primers F2 and F3 corresponding to
exon 2 and also exon 3, but further 5’ than primer F4,
amplified fragments of 263 bp, still including intron 2 at the
DNA level and 74 bp at the RNA level (Fig 1). Preincubations of the RNA template isolated from Hep G2 cells with
RNAase-free DNAase I or DNAase-free RNAase A were
followed by disappearance of either the DNA- or the
RNA-specific band after enzymatic amplification (Fig 2).
Restriction enzyme digestion with the enzyme DdeI, which
cuts at position 2OO2,I4generated characteristic fragments
of 242 bp from DNA templates and 53 bp from RNA
templates (Fig 2).
Megakaryocytes, Hep G2 cells, and fibroblasts were
tested with the previously mentioned PCR (Fig 1). A
DNA-specific band could be detected in all three samples.
Neither DNA-specific nor RNA-specific bands were detected when primers without template or pure yeast tRNA
(data not shown) were used as template. An RNA-specific
band was present only in Hep G2 cells.
For GPIIIa a second nested primer PCR could be
established. Again, a similar approach as for the fibrinogen
y-chain was used. The external set of primers corresponded
to exon 1 for primer G1 and exon 5 for primer G4, the
internal set with primer G2 to exon 2 and primer G3 to exon
4. Due to the yet unknown exact length of the intervening
introns the distance between both sets of primers at the
DNA level can only be estimated to be considerably longer
than 5 kb. No bands of a similar length were detected in any
of the tested cells. However, at the RNA level fragments of
1,086 bp with the external primers and of 303 bp with the
internal set of primers were generated (Fig 3). Diagnostic
fragments of 178 bp and 125 bp (Fig 3) resulted from Bum
HI restriction enzyme digestion of the 303-bp PCR product
at position 1496.’’
Of all analyzed samples only megakaryocytes showed
expression of GPIIIa mRNA. Hep G2 cells and fibroblasts
remained negative as well as pure yeast tRNA (data not
shown) and primers without RNA template (Fig 3).
The housekeeping gene aldolase A served as the positive
control for all tested cells. The primer sequence was
published by Chelly et al.” A 181-bp fragment could be
amplified by primers H, and AZ5,which is specific for
aldolase A mRNA. In all cases with negative PCR results
for GPIIIa or y-chain fibrinogen mRNAs, the aldolase A
PCR showed the characteristic band of 181 bp when an
aliquot of the same RNA preparation was used as template.
The aldolase A PCR was also negative when primers
without RNA template or pure yeast tRNA as template
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LANGE ET AL
22
Fig 1. Ethidium bromldestained agarose gel of fibrinogen
ychain PCR products amplified
from different RNA sources. MW,
dIX174/Hae 111; lane 1, enriched
megakaryocytes; lane 2, Hop G2
cells; lane 3, MRC 5 fibroblasts;
lane 4, primers without template; lane 5, Hop G2 PCR product/Ddel. Characteristic fragments are of 263-bp length for
DNA templates and 74 bp for
RNA templates. In Ddel digests
the diagnostic fragments are of
242 bp (DNA) and 53 bp (RNA);
the short 21-bp fragment is lost
from this gel (see Fig 2).
310-
72-
1
MW
2
3
4
wcrc uscd (Fig4). By this control, PCR ncgativc rcsults duc
to degraded RNA tcmplatcs could bc cxcludcd.
Thc rcsults of thc PCR analyses from thc diffcrcnt RNA
sourccs arc summarizcd in Tablc 1.
DISCUSSION
To study whcthcr human mcgakaryocytc/platclct fibrinogen is synthcsizcd by megakaryocytcs or aquircd from thc
plasma and delivered to a-granulcs, wc established a highly
5
scnsitivc ncstcd primer PCR for thc dctcction of human
fibrinogcn y-chain mRNA. Thc y-chain of fibrinogen is of
utmost importance for hemostasis and platclct aggregation;
it interacts with platclct GPllb-llla through a dodccapcptidc located at thc carboxy tcrminus' and providcs binding
sitcs for FXllla-mcdiatcd polymcrization of fibrin.' It
occurs in thrcc forms in human plasma; thc smallcr y-50
protein (T-Val 41 1) and thc largcr y-57.5 protein (T -Lcu
427) arc produced by altcrnativc splicing; thc intcrmcdiatc
310Fig 2. Ethidium bromidestained agarose gel with fibrinogen +sin
PCR productsamplified from Hep G2 cells. MW,
dIX174/Hae 111; lane 1, Hap G2
PCR products; lane 2, Ddel restriction e n q m e digest of the
DNA- and RNA-specific bands;
lane 3, PCR performed after
RNAse pretreatment of RNA template; lane 4, PCR performed after DNAase preincubation of RNA
template. The correct-sized PCR
products of 263 bp for DNA and
74 bp for RNA are present (lane
1). After restriction enzyme digestion, diagnostic fragments of 242
bp and 21 bp for DNA templates
and 53 bp and 21 bp for RNA
templates are shown (lane 2).
-263
-24
-74
-53
72-
-21
MW
1
2
3
4
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23
LACK OF FIBRINOGEN EXPRESSION IN MEGAKARYOCYTES
Fig 3. Ethidium bromidestained agarose gel for GPllla
PCR products. M W , bX174lH.m
111; lane 1, enriched megakaryocytes; lane 2, Hep G2 cells; lane
3, MRC 5 fibroblasts; lane 4, primers without template; lane 5.
Bsm HI restrlction enzyme digest
of the PCR product amplified
from the megakaryocyte RNA
template. The RNA-specific band
has a size of 303 bp. After Bsm HI
digestion diagnostic fragments
of 178 bp and 125 bp are present.
1
b1
310194-
-303
!I
-178
,I
118-
-125
1
y-55 protein (I. -Pro1 423) is a posttranslational moditication of the y-57.5
Thc heterogeneity is due to
variations in the amino acid scqucncc at the C-terminal
end. Therefore, wc chose a scqucncc located near the
5'-cnd of the fibrinogen y-chain gene that is identical in all
fornis of y-fibrinogens for an c n q m a t i c amplification o f
tibrinogcn y-chain DNA and RNA.
Primers wcrc constructed in a way that not only exon
scqucnccs. but also intcrvcning intron scqucnccs wcrc
ampliticd. Due to this fact i t was possible to distinguish
2
3
4
5
unequivocally fragnicnts originating from DNA or from
RNA templates. An cxtcnsivc homology scarch was pcrformed to exclude niispriming due to scqucncc homologics
bctwccn the different fibrinogen a-,p-. and y-chains during
the PCR. To further incrcasc the sensitivity and spccificity
we chose the nested primer approach in which a second set
of internal prinicrs is used in a subsequent second round of
PCK. 'The faithful amplification could be contirmcd by the
correct siLc of the PCK products and in restriction c n q m c
digests that generated thc expected-sized fragments. By
310-
194-
34
k
MW
1
2
3
4
-181
Fig 4. Aldolase A PCR products stained with ethidium bromide after agarose gel electrophoresis. MW, &X174/Hee 111;
lane 1. enrlchedmegakaryocytes;
lane 2, Hep G2 cells; lane 3, MRC
5 fibroblasts; lane 4, primers
without template.
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24
LANGE ET AL
Table 1. Summarized Results of PCR Analyses From
Different RNA Sources
?-Fibrinogen
Megakaryocytes
Fibroblasts
Hep G2
GPllla
RNA
Aldolase A
RNA
-
+
+
-
-
f
-
+
DNA
RNA
+
+
+
+
pretreatment of the mRNA template with RNAase or
DNAase it was possible to amplify only DNA- or RNAspecific fragments.
Based on the data and primer sequences of Newman et
the same kind of nested primer PCR could be established for GPIIIa mRNA that encodes for the GPIIIa
component of the GPIIb/IIIa receptor on megakaryocytes
and platelets. This reaction served as the positive control
for megakaryocyte-specific mRNA. Due to rather long
intervening intron sequences only GPIIIa mRNA and not
DNA could be amplified; therefore, RNA templates underwent no DNAase pretreatment. The amplified fragments
were of the correct size and showed the expected bands
after BamHI restriction enzyme digestion. After RNAase
treatment no bands could be amplified.
As a positive control for the presence of intact mRNA in
all preparations, RNA-specific sequences of the housekeeping gene aldolase A were amplified. Only experiments in
which a usual, not nested, primer PCR for aldolase A was
positive after 30 cycles were evaluated. Therefore, negative
results due to degraded RNA could be excluded.
Three different types of cells, the hepatoma cell line
HepG2, the fibroblast cell line MRCS, and megakaryocytes
from healthy volunteers were tested for fibrinogen y-chain
mRNA expression. In our PCR systems megakaryocytes
were only positive for GPIIIa and aldolase A mRNA
expression. We were unable to find fibrinogen y-chain
mRNA in megakaryocytes as detectable by our highly
sensitive nested primer PCR technique, whereas HepG2
cells were clearly positive. The most likely explanation for
this finding is that fibrinogen y-chain mRNA and fibrinogen
are not synthesized in megakaryocytes. As PCR can fail
with homologies only slightly below loo%, the possibility
that the megakaryocyte mRNA is a different gene product
as compared with the fibrinogen y-chain mRNA of the
HepG2 cells cannot be excluded with absolute certainty.
The fact that the DNA-characteristic band for the
fibrinogen y-chain gene could be amplified from megakaryocytes, HepG2 cells, and fibroblasts indicates that the
RNA preparation method does not yield DNA-free RNA.
Residual DNA of the cultured cells or the freshly prepared
megakaryocytes is probably responsible for such a contaminant; this was proven by preincubation of the RNA template with DNAse I, after which a DNA-specific band was
no longer detectable.
Our results are in contrast to observations in guinea pig
megakaryocytes" and human megakaryocytes? where fibrinogen biosynthesis was shown by "S-methionine labeling;
moreover, y-chain mRNA was identified by Northern
blotting in rat megakaryocytes by Uzan et a1.22However,
other data7.8x23
indicate that megakaryocyteiplatelet fibrinogen might be of exogenous origin, being taken up from the
surrounding plasma, as demonstrated by the incorporation
of circulating IgGZ4as well as the exogenous tracer protein
HRPz into a-granules of megakaryocytes. In vitro studies
with cultures of human megakaryocytes showed that fibrinogen was only observed in a-granules when an exogenous
source of fibrinogen was present in the culture system.'
Moreover, Harrison et al* have provided in vivo evidence
for the endocytic uptake of plasma fibrinogen into human
megakaryocytes and platelet a-granules in a patient with
congenital afibrinogenemia, given replacement therapy with
cryoprecipitate. The mechanism of megakaryocyteiplatelet
endocytosis of fibrinogen remains unknown at present.
In platelets the y-57.5 form of the y-chain is absent or
almost completely reduced in a-granulesZ6;these differences between platelets and plasma fibrinogen have long
been used to argue against the uptake of fibrinogen from
the surrounding milieu. However, there might be a preferential uptake of 7-50 from plasma as compared with the
y-57.5 protein, or the differences are caused by platelet
proteases."
Considering a coordinate regulation of the a, p, and y
genes at the transcriptional level,28our results suggest that
human megakaryocytes do not synthesize fibrinogen. This
protein might enter megakaryocytesiplatelets and get incorporated and concentrated in a-granules and stored for later
secretion. The storage of plasma fibrinogen in megakaryocytes/platelets might ensure the supply of fibrinogen at sites
where platelets are the first cells to adhere to damaged
vascular structures, thus providing the delivery of high
concentrations of this molecule at the sites of primary
hemostasis.
NOTE ADDED IN PROOF
Published subsequent to submission of this manuscript, Louache
et a1 have drawn similar conclusions based on mRNA for fibrinogen a-and P-chains (Blood 77:311, 1991).
ACKNOWLEDGMENT
The authors thank Dagmar Wider for excellent technical assistance.
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LACK OF FIBRINOGEN EXPRESSION IN MEGAKARYOCYTES
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1991 78: 20-25
Fibrinogen gamma-chain mRNA is not detected in human
megakaryocytes
W Lange, A Luig, G Dolken, R Mertelsmann and L Kanz
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